The normal development of B lymphocytes results in the production, on the surface of mature B lymphocytes, of immunoglobulins with isotypes M and G, initially having common isotypes. Plasmocytic disorders which are at the origin of monoclonal diseases result from a control default in the process of cell maturation into antibody secreting cells after exposure to a specific antigen of the surface immunoglobulin. In that situation, immunoglobulins having an affinity for an antigen to which the host has been exposed continue to be secreted after the disappearance of the antigen. In general, immunoglobulins of a given species are distributed into different classes of antibodies, each class being identified by an isotype, the different isotypes identified within a species being common to all normal individuals of that species. Immunoglobulins are generally formed from heavy chains (2 heavy chains) and light chains (2 light chains). Five heavy chain isotypes (M, G, A, D, E) and two light chain isotypes (kappa and lambda) have been identified in that four-chain structure. In addition to their isotype, immunoglobulins are characterized by determinants corresponding to differences between the individuals of a given species, termed allotypes, as well as by their idiotype, corresponding to a portion of the immunoglobulin molecule binding the antigen. Thus, the idiotype is characteristic of molecules produced by a given clone of antibody producing cells.
Plasmocytic diseases are thus particularly characterized by an alteration in the production of certain immunoglobulins or certain immunoglobulin chains, and detection and characterization of that alteration is of great clinical importance.
Electrophoretic analysis of a biological sample allows identification of seric proteins and determination of the quantity of those proteins, in particular of immunoglobulins. In an electric field, proteins migrate as a function of their size and charge, forming an electrophoresis profile comprising a series of peaks (also termed fractions), each corresponding to one or more proteins. The gamma fraction is especially formed by immunoglobulins, principally type G. In patients suffering from plasmocytic diseases leading to the secretion of monoclonal proteins (also termed Mc proteins), the quantity of immunoglobulins corresponding to one of the known isotypes may be significantly increased over the normal quantity, leading to a modification in the electrophoretic profile by modification of one or more peaks for the proteins separated from a serum sample.
The detection of monoclonal proteins, in particular the detection of an increase in the production of a particular immunoglobulin isotype, is thus of great importance when investigating plasmocytic diseases, but also when monitoring patients with paraproteinemias. As an example, it has been observed that, depending on the case and in particular during tumor development, the quantity of a seric Mc protein can be directly related to the progress of the disease. A Mc protein may thus constitute a tumoral marker which, when correlated with other symptoms, can be taken into account when making a diagnosis.
Plasmocytic diseases are not only concerned with the abnormal production of Mc proteins; pathological disorders associated with the production of Mc proteins that can be cited include lymphoid neoplasms, such as chronic lymphoid leukemia or lymphomas of B or T lymphocytic origin, certain non lymphoid proliferations such as chronic myeloid leukemia, and cancers of the breast or colon.
Monoclonal Mc proteins can also be produced in certain non malignant diseases such as cyrrhosis, sarcoidosis, parasitoses or Gaucher's disease. The production of monoclonal proteins is also detected in autoimmune diseases such as rheumatoid polyarthritis, myasthenia, or cold agglutinin disease.
By enabling detection of the presence of monoclonal proteins in biological samples and following the change in the detected monoclonal proteins with time, agarose gel and capillary electrophoresis, thus constitute the methods of choice with a view to establishing a diagnosis or monitoring patients.
Depending on the diseases concerned, the Mc protein is of a different nature, constituted either by an intact antibody molecule, or by a fragment of antibody. Thus, heavy chains or light chains can be produced alone. This is the case, for example, with Bence Jones proteins secreted in the urine of patients with myelomas, which are in the form of light chains alone.
The isotypes that are determined for the immunoglobulins allow the Mc proteins to be typed as a function of the nature of their heavy chain and/or as a function of the nature of their light chain. The technique used to type immunoglobulins thus allows the type of heavy and/or light chains associated with each monoclonal protein in a biological sample to be determined.
In addition to detecting the presence of said Mc proteins, it thus appears important to type them to enable the disease associated with them to be characterized. To this end, different approaches have already been proposed, such as column chromatography, agarose gel electrophoresis or capillary electrophoresis. In methods employing agarose gel electrophoresis, proteins from the biological sample are separated by electrophoresis in the form of an electrophoresis profile in which the proteins and in particular the globulins are revealed in the form of peaks or bands that may include a monoclonal protein, the nature of which must then be confirmed, for example by immunofixation on agarose gel. That technique combines two steps by using agarose gel electrophoresis then immmunoprecipitation. Several aliquots of the same biological sample are deposited in parallel on agarose gel, then an electric field is applied to separate the proteins, in particular the immunoglobulins. Each track is then incubated with a type of antibody that is specific to the types of immunoglobulins being investigated (IgG, IgA, IgM, kappa and lambda, and possibly free kappa, free lambda, IgD and IgE), leading to the formation of immunocomplexes between the immunoglobulins in the sample and the antibodies. After washing the gel to eliminate non-precipitated proteins, a staining step reveals the position of the immunocomplexes: in the absence of monoclonal proteins, only a diffuse stained background appears (corresponding to a multitude of monoclonal antibodies constituting the “polyclonal background”); in the presence of monoclonal proteins, stained bands are revealed in specific regions of the gel. Using a reference track (no antiserum), each monoclonal band that is visible on the gel can then be typed.
One typing technique used in capillary electrophoresis is immunosubtraction, as described in U.S. Pat. No. 5,228,960. That technique consists of incubating aliquots of a biological sample with specific antibodies that can subtract a given constituent (for example immunoglobulins) from said sample. That constituent remains adsorbed on the solid phase on which the antibody is fixed: thus, it is no longer present in the sample analyzed by capillary electrophoresis. The different treated aliquots from which certain immunoglobulins have been subtracted depending on the specificity of the antibodies used are then injected into the capillary, then the proteins contained in the aliquots are separated by applying an electric field to the terminals of the capillary. The profiles obtained are compared with an untreated aliquot profile.
Other techniques for identifying monoclonal proteins separated by capillary electrophoresis from a sample have been proposed, for example in European patent EP-B1-0 690 988. That European patent describes the combination of capillary electrophoretic separation with an immunosubtraction step with the aim of facilitating the classification of Mc proteins. In that patent, immunosubtraction is carried out “on-capillary” using a method which comprises a step for electrophoretic separation of a first portion of the sample into its different constituents, and detection of those constituents, then mixing a second portion of the sample with at least one partner that is capable of specifically binding a predetermined analyte contained in the sample, it being understood that the specific partner for binding has an electrophoretic mobility that differs from that of said analyte. The second portion of the sample is then separated into its different constituents by capillary electrophoresis and the constituents are detected. A step for comparing the separated constituents is then carried out with the constituents separated without the presence of specific partners binding a desired analyte.
EP-B1-0 690 988 states that the partner intended to bind the analytes is a modified molecule, in particular an anti-Mc protein antibody, which has undergone chemical modification so that its migration time in capillary electrophoresis takes it outside the gamma region of the electrophoretic profile. One chemical modification that is proposed for the antibodies is that which consists of reacting them with succinic anhydride to provide them with additional carboxylic functions, negative at alkaline pH. Under the analytical conditions described (pH 10), the overall negative charge of the antibodies is thus increased.
The conditions described for modifying the antibodies in EP-A-0 690 988 and the experimental results that are reported, which solely concern the analysis and separation of purified G immunoglobulins and not serum analysis, throw doubt on the pertinence of the proposed method when investigating monoclonal proteins in a “real” biological sample, for example in a serum.